INHALED ANESTHETIC BINDING INTERACTIONS

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The long term goals of the project are 1) to understand interactions responsible for the binding of inhaled anesthetics, which are small hydrophobic molecules, to target proteins and lipids; 2) to identify structural motifs for favored binding sites in these targets; and 3) to determine structural consequences of inhaled anesthetic binding to macromolecular targets as a first step towards understand functional consequences. The project will use a direct photoaffinity labeling approach recently developed and validated by the PI to provide quantitative anesthetic binding data in a variety of models chosen for their simplicity and predictability. Aside from defining features of anesthetic binding sites, these quantitative binding data will be used to set initial conditions in dynamic simulations, and will also be essential for reconciling results of other approaches in this program. There are four specific aims to accomplish short term objectives, which characterize how and where inhalational anesthetics bind to model peptides and lipids. Specific aim 1 is to determine the minimum structural requirements for binding selectivity of halothane and other inhalational anesthetics to peptides. Homopolymers of poly-(L-lysine) will be studies where there are known secondary structural features and manipulatable supersecondary structures. These hompolymers will serve as a scaffold for the inclusion of other amino acids. Specific aim 2 is to determine anesthetic binding character and location in synthetic helical bundles of known structure. Water soluble helical bundles synthesized in Project II will be photo labeled to determine halothane binding locations, specificities, and affinities. Systematic amino acid substitutions at selected positions will allow correlation of cavity features with anesthetic binding. Specific aim 3 is to determine the character and location of halothane that is photochemically incorporated into lipid bilayers of varying composition. In this aim the project will interact with Project IV regarding the initial placement of halothane and the effect of physical composition and changes. Specific Aim 4 will determine the importance of lipid/protein interfaces regarding specific and functional relevant anesthetic binding domains. Binding character and location will be correlated with function in planar bilayers in selected membrane-incorporated synthetic helicies. This aim strongly interacts with Project IV.